JP2018072094A - Method and device of embryo inspection of fish - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an embryo inspection method of a zebra fish capable of realizing a practically usable inspection accuracy and inspection time.SOLUTION: An excitation light source 3 is provided obliquely above a fish egg tray 2 arranged with a number of wells 20 accommodating one embryo 10 of a zebra fish together with the water, and a fluorescence detector 4 is provided immediately above the fish embryo tray 2. A highly accurate determination between a normal egg and an abnormal egg can be made based on an optical spectrum intensity of a peak wavelength or a narrow channel alone in the vicinity thereof from among the fluorescence spectra acquired from the embryo of the zebra fish after five to eight hours elapsed from the conception using an inspection device for inputting a signal voltage outputted from the fluorescence detector 4 into a determination part 5 for the embryo. The high-speed and high accuracy quality determination can be realized using a multi-well plate.SELECTED DRAWING: Figure 1

Description

The present invention relates to an inspection method capable of determining whether a fertilized egg of a fish such as a zebrafish is good and accurate in a short time.

Zebrafish has the advantage that it lays eggs almost every day and has a very short growth period until it becomes an adult fish. Furthermore, since the zebrafish is small in size, it is easy to handle, and there is an advantage that a large number of data can be easily obtained with a relatively small device. For this reason, zebrafish are attracting attention as experimental materials in fields such as genetic research, pathological research, and pharmaceutical research. For example, a zebrafish utilization method has been proposed in which a genetic material is injected into a fertilized egg of a zebrafish to recover a target substance derived from the genetic material from an adult fish. Also, a zebrafish utilization method has been proposed in which a biological effect is evaluated by introducing a drug or the like into a fertilized egg of zebrafish.

Conventionally, optical methods using visible light, infrared light, ultraviolet light, and the like have been used as noninvasive observation methods for living tissue. For example, Patent Document 1 proposes determining the quality of a fertilized egg using a visible light spectrum or an infrared light spectrum. Patent Document 2 proposes to detect the characteristics based on a fluorescence spectrum from a living tissue. Patent Document 3 proposes to measure fluorescence of a large number of cells dispersedly arranged in a multiwell plate manufactured using a cycloolefin polymer which is a low fluorescent material.

Special table 2004-516475 gazette JP-T-2004-518124 Special Table 2002-515125

The current zebrafish technology has a problem that the ratio of zebrafish that normally hatch from fertilized eggs is considerably low. This is because a considerable number of the fertilized eggs of zebrafish obtained become defective eggs such as dead eggs and weak eggs.

If these dead and weak eggs are not eliminated immediately, there is a possibility of contaminating the water system. As a result, other normal eggs may be adversely affected through this contaminated water. Once an aqueous system containing a large number of zebrafish fertilized eggs is once contaminated with pathogenic bacteria, viruses, or other substances, they attach to the surface of normal eggs, which has serious consequences.

In addition, when a drug or the like is injected into a fertilized egg before hatching, it is not economical to consume expensive test materials for defective or abnormal eggs such as dead eggs or weak eggs. In addition, it was impossible to determine whether the abnormality of zebrafish hatched from a defective egg or an abnormal egg was due to drug injection or the original one. Furthermore, injection into a defective egg infected with bacteria or viruses may cause infection of other normal eggs through the injection needle.

Eventually, it was found that in the industrial use of zebrafish, it is necessary to select and use only normal fertilized eggs in a short time from a large number of zebrafish fertilized eggs. However, considering that the cell division rate of a zebrafish fertilized egg is high and the number of fertilized eggs to be examined is very large, the examination time allowed per fertilized egg needs to be extremely short.

However, for example, a method for selecting a fertilized egg of a very small zebrafish with a diameter of about 1 mm in a short time has not yet been reported. From these viewpoints, the inventors have realized that in order to realize the industrial use of zebrafish, it is essential for the industrial use of zebrafish to establish a highly accurate and high-speed fertilized egg inspection method. .

Fluorescence spectrum analysis technology is promising for testing fertilized eggs in zebrafish because it is a non-invasive testing method. However, it has been unclear whether a high-speed and high-precision zebrafish fertilized egg test can be realized using a known fluorescence spectrum analysis technique. Furthermore, even if possible, the specific method for realizing the determination with high accuracy was unknown. In particular, the specific method for completing a large number of fertilized eggs of zebrafish in a short time was unknown. In other words, no fluorescence spectrum analysis technique capable of realizing the required inspection accuracy and inspection speed has been known.

The present invention has been made in view of the above problems required for fertilized eggs of zebrafish, and is to provide an optical inspection method capable of discriminating between good and defective zebrafish fertilized eggs with high accuracy and high speed.

According to the present invention, among the fluorescence spectra obtained from fertilized eggs of zebrafish, the spectrum of excitation light or a very narrow band adjacent thereto is adopted as the spectrum for inspection. According to the observations of the present inventors, it is found that the spectrum for examination of normal eggs is relatively narrow band or low intensity compared to that of dead eggs or weak eggs (hereinafter referred to as bad eggs). It was.

In one example, the narrow spectrum inspection spectrum adjacent to the excitation light spectrum was much less intense in normal eggs than in bad eggs. In another example, the inspection spectrum that substantially overlaps the band of the excitation light spectrum was much lower in the normal egg than in the defective egg. Furthermore, when the narrow-band inspection spectrum adjacent to the short wavelength side of the excitation light spectrum is optimal according to the wavelength of the excitation light, the narrow-band inspection spectrum adjacent to the long wavelength side of the excitation light spectrum is In the optimal case, it was found that a narrow spectrum inspection spectrum that overlaps the excitation light spectrum may be optimal.

In another example, in determining the inspection spectrum adjacent to the peak wavelength spectrum of a normal egg, the pass / fail of the fertilized egg is determined based on the bandwidth of the total peak wavelength spectrum of the peak wavelength spectrum and the inspection spectrum. You can also That is, if the bandwidth of the total peak wavelength spectrum exceeds a predetermined threshold value, it is determined as a defective egg.

In another example, it is determined whether or not the intensity of the spectral spectrum in a band less than 30 nm away from the excitation light band in the fluorescence spectrum of the fertilized egg exceeds a predetermined threshold value, and if so, the fertilized egg is defective. Can be determined.

In another example, it is determined whether or not the intensity of the spectral spectrum for examination that substantially overlaps the excitation light band in the fluorescence spectrum of the fertilized egg exceeds a predetermined threshold value, and if so, the fertilized egg is determined to be defective. can do.

Furthermore, the present inventors have found that the above-described difference in the intensity of the spectral spectrum for inspection between normal and bad eggs has passed 5-8 hours, more preferably 5.5-7.5 hours from the time of fertilization. It was discovered that the accuracy was sufficiently high at the time of observation. Therefore, by performing selection at this observation time, it becomes possible to eliminate defective eggs before the eggshells of fertilized eggs are destroyed by hatching.

According to a preferred embodiment, in order to inspect a large number of fertilized eggs of zebrafish at high speed, a multi-well plate in which a large number of wells for accommodating fertilized eggs are formed on a bottom plate is driven horizontally. A two-dimensional address of a well containing a fertilized egg determined to be defective in the inspection is stored. After the inspection is completed, fertilized eggs with defective addresses are sequentially removed. Thereby, the test | inspection of many fertilized eggs can be completed in a short time. The multi-well plate formed on the bottom plate of the well preferably has a state in which the fertilized egg is stably accommodated and does not move, and preferably has a cylindrical or polygonal cylindrical bottom portion as close as possible to the diameter of the fertilized egg.

It is a block diagram which shows the test | inspection apparatus which enforces the fertilized egg test | inspection method of the zebrafish of an Example. It is a figure which shows the fluorescence spectrum of a normal egg and an abnormal egg. It is a figure which shows the fluorescence spectrum of a normal egg and an abnormal egg. It is a figure which shows the fluorescence spectrum of a normal egg and an abnormal egg. It is a figure which shows the fluorescence spectrum of a normal egg and an abnormal egg. It is a figure which shows the fluorescence spectrum of a normal egg and an abnormal egg. It is a figure which shows the fluorescence spectrum of a normal egg and an abnormal egg. It is a figure which shows the fluorescence spectrum of a normal egg and an abnormal egg. It is a figure which shows the fluorescence spectrum of a normal egg and an abnormal egg.

FIG. 1 is a schematic block diagram showing a zebrafish fertilized egg inspection apparatus. A fish egg tray 2 is placed on a two-dimensional movable XY table 1. However, in FIG. 1, the fish egg tray 2 is schematically illustrated.

An excitation light source 3 is provided obliquely above the fish egg tray 2. A fluorescence detector 4 is provided immediately above the fish egg tray 2. The signal voltage output from the fluorescence detector 4 is input to the determination unit 5 for determining a fertilized egg.

The XY table 1 is movable in a horizontal X direction and a right angle and a horizontal Y direction by two stepping motors (not shown). The fish egg tray 2 has a large number of wells 20 constituted by recesses whose upper surfaces are opened. Each well 20 is arranged in a matrix. Each well 20 contains one fertilized egg 10 of zebrafish with water. The fish egg tray 2 is formed using a low fluorescence induction resin material, but is not limited thereto.

The excitation light source 3 is constituted by a laser having a predetermined wavelength. The excitation light source 3 emits laser light 30 in a direction of about 45 degrees with respect to the horizontal X direction. The excitation light source 3 incorporates a lens system for emitting laser light in parallel to almost the entire inspection well 20A, which is the well 20 at the inspection position. You may employ | adopt the spectrum of the predetermined wavelength obtained with the diffraction grating from the xenon lamp as the excitation light source 3. FIG.

The fluorescence detector 4 detects a predetermined direction component 40 of the fluorescence spectrum emitted from the inspection well 20A and converts it into a signal voltage. The optical axis of the fluorescence detector 4 that coincides with the predetermined direction component 40 extends in a direction of about 45 degrees with respect to the horizontal Y direction. Therefore, the angle between the optical axis of the excitation light source 3 and the optical axis of the fluorescence detector 4 is 90 degrees. As a result, the amount of the excitation light emitted from the excitation light source 3 entering the fluorescence detector 4 is reduced. The fluorescence detector 4 includes a lens system having a focus on the inspection well 20A, a prism or diffraction grating that divides a substantially parallel fluorescence beam emitted from the lens system, and a spectral spectrum of a predetermined band emitted from the prism or diffraction grating. At least a photoelectric sensor for photoelectric conversion. Optical elements such as slits for narrowing the beam width can also be added.

In other words, this photoelectric sensor is disposed at a position where only a spectral spectrum of a specific wavelength is incident among fluorescent spectral spectra emitted from a prism or a diffraction grating. Thereby, the fluorescence detector 4 can detect only a predetermined narrow-band fluorescence spectrum.

The signal voltage output from the photoelectric sensor is converted into a digital signal by an A / D converter and input to the microcomputer 5. The microcomputer 5 determines the quality of the zebrafish fertilized egg in the test well 20A based on the input digital signal, and controls intermittent light emission of the excitation light source 3 and intermittent movement of the XY table 1. Thereby, the quality of the fertilized egg of the zebrafish in each well 20 is determined sequentially. FIG. 1 shows a basic configuration of an inspection apparatus, and it is needless to say that various modifications can be made using a known technique.

Next, observation examples of fluorescence spectra obtained from normal and abnormal fertilized eggs of zebrafish will be described. The dotted line shows the fluorescence spectrum of a normal egg, and the solid line shows the fluorescence spectrum of an abnormal egg that is a dead egg. In addition, it turned out that the fluorescence spectrum from the weak egg which cannot complete hatching in the same time slot | zone as a normal egg has the intermediate value of a dead egg and a normal egg in the zone | band of the spectrum for a test | inspection.

First Example FIG. 2 shows a fluorescence spectrum obtained by irradiating a fertilized egg 6 hours after fertilization with excitation light having a wavelength of 310-330 nm. It was found that the intensity difference of the fluorescence spectrum between the abnormal egg and the normal egg exists in the wavelength band of 280-300 nm. For example, at a wavelength of 280 nm, the fluorescence relative intensity value of dead eggs exceeded 10, whereas that of normal eggs was less than 2. Therefore, for example, if the classification threshold is the fluorescence relative intensity value 6, dead eggs including weak eggs can be discriminated with high accuracy.

Second Example FIG. 3 shows a fluorescence spectrum obtained by irradiating a fertilized egg 7 hours after fertilization with excitation light having a wavelength of 310-330 nm. The result was almost the same as the first example.

Third Example FIG. 4 shows a fluorescence spectrum obtained by irradiating a fertilized egg 6 hours after fertilization with excitation light having a wavelength of 390-410 nm. It was found that the difference in the intensity of the fluorescence spectrum between the abnormal egg and the normal egg exists in the band of wavelength 360-380 nm. For example, at a wavelength of 370 nm, the fluorescence relative intensity value of dead eggs exceeded 10, whereas that of normal eggs was less than 2. Therefore, for example, if the classification threshold is the fluorescence relative intensity value 6, dead eggs including weak eggs can be discriminated with high accuracy.

Fourth Example FIG. 5 shows a fluorescence spectrum obtained by irradiating a fertilized egg 7 hours after fertilization with excitation light having a wavelength of 390-410 nm. The result was almost the same as the third example.

FIG. 6 shows a fluorescence spectrum obtained by irradiating a fertilized egg 6 hours after fertilization with excitation light having a wavelength of 590-610 nm. It was found that the difference in the intensity of the fluorescence spectrum between the abnormal egg and the normal egg exists in the wavelength band of 600-620 nm. For example, at a wavelength of 610 nm, the fluorescence relative intensity value of dead eggs exceeded 10, whereas that of normal eggs was less than 2. Therefore, for example, if the classification threshold is the fluorescence relative intensity value 6, dead eggs including weak eggs can be discriminated with high accuracy.

FIG. 7 shows a fluorescence spectrum obtained by irradiating a fertilized egg 7 hours after fertilization with excitation light having a wavelength of 590-610 nm. The result was almost the same as the fifth example.

FIG. 8 shows a fluorescence spectrum obtained by irradiating a fertilized egg 6 hours after fertilization with excitation light having a wavelength of 790-810 nm. It was found that the intensity difference of the fluorescence spectrum between the abnormal egg and the normal egg exists in the wavelength band of 780-820 nm. For example, at a wavelength of 790 nm, the fluorescence relative intensity value of dead eggs exceeded 10, but that of normal eggs was almost 2. Therefore, for example, if the classification threshold is the fluorescence relative intensity value 6, dead eggs including weak eggs can be discriminated with high accuracy.

Eighth Example FIG. 9 shows a fluorescence spectrum obtained by irradiating a fertilized egg 7 hours after fertilization with excitation light having a wavelength of 790-810 nm. In the wavelength band of 780 to 820 nm, which is the same as the excitation light, the fluorescence relative intensity value of dead eggs exceeded 10, while that of normal eggs was less than 4. Therefore, for example, if the classification threshold is the fluorescence relative intensity value 7, dead eggs including weak eggs can be discriminated with high accuracy.

These observations revealed the following. First, it was found that the peak wavelength of the fluorescence spectrum emitted from an abnormal egg was almost constant regardless of the individual difference of fertilized eggs or the elapsed time from fertilization. Therefore, it was found that the quality determination of fertilized eggs of zebrafish can be realized with the highest accuracy by selectively detecting only the fluorescence spectrum of a very narrow band depending on the excitation light wavelength.

Next, when the excitation light wavelength is short, in the narrow band adjacent to the short wavelength side of the excitation light wavelength, the intensity of the fluorescence spectrum of the abnormal egg is significantly increased compared to that of the normal egg. As the excitation light wavelength becomes longer, the intensity of the fluorescence spectrum of the abnormal egg is significantly increased compared to that of the normal egg in a narrow band adjacent to the longer wavelength side of the excitation light wavelength. As the excitation light wavelength becomes longer, the intensity of the fluorescence spectrum of the abnormal egg in the same band as that of the excitation light greatly increases compared to that of the normal egg. Furthermore, the intensity difference in the fluorescence spectrum between the normal egg and the abnormal egg increases with the elapsed time from fertilization, but when the elapsed time exceeds 8 hours, the intensity difference is almost saturated or decreased. I found out that Furthermore, it has been found that sufficient discrimination accuracy can be obtained when the elapsed time from the time of fertilization is in the range of 5-7 hours, more preferably 5.5-6.5 hours.
In the above description, the zebrafish has been mainly described. However, the present invention is not limited to this, and can be applied to fishes although the spectral spectrum position is slightly different. Moreover, reflected light may be used instead of fluorescence, and the ratio of reflected light included in fluorescence may be increased.

Claims (8)

A fertilized egg inspection method for optically inspecting fish fertilized eggs,
A fertilized egg test for fish, wherein the quality of the fertilized egg is determined based on a spectral spectrum for inspection consisting of a spectrum of excitation light or a narrow-band spectral spectrum adjacent to the fluorescent spectrum obtained from the fertilized egg. Method.   In the fluorescence spectrum obtained from the fertilized egg, it is determined whether or not the intensity of the spectral spectrum for inspection including a spectral spectrum in a band separated by less than 30 nm from the excitation light band exceeds a predetermined threshold value. The method for inspecting fish fertilized eggs according to claim 1, wherein it is determined that the fish is defective.   It is determined whether or not the intensity of the inspection spectrum including the spectrum in the excitation light band out of the fluorescence spectrum obtained from the fertilized egg exceeds a predetermined threshold value, and if so, the fertilized egg is determined to be defective. The method for inspecting fish fertilized eggs according to claim 1.   2. The fish fertilized egg inspection method according to claim 1, wherein the spectroscopic spectrum for inspection is detected at an observation time when 5 to 8 hours have elapsed from the time of fertilization.   The fish fertilized egg inspection method according to claim 4, wherein the spectroscopic spectrum for inspection is detected at an observation time point 5.5 to 7.5 hours after the fertilization time point. In each well formed on the upper surface of the bottom plate of the multiwell plate, fertilized eggs of fish are stored together with water,
After aligning the center of the well with the optical axis of the excitation light, by irradiating the fertilized egg in the well with the excitation light, a predetermined spectral spectrum of the fluorescence spectrum obtained from the fertilized egg is detected,
After determining the quality of the fertilized egg based on the spectral spectrum, by horizontally moving the multi-well plate, the same detection operation is sequentially repeated for the fertilized eggs of the adjacent wells,
The fish fertilized egg inspection method according to any one of claims 1 to 4, wherein pairs of the obtained determination result for each well and the two-dimensional address of each well are sequentially stored. Each well formed on the upper surface of the bottom plate of a multi-well plate that contains fertilized eggs of fish together with water,
A light source that is incident with the center of the well aligned with the optical axis of the excitation light; and a light receiver that receives the reflected light obtained from the fertilized egg by irradiating the fertilized egg in the well with excitation light;
A measuring device that measures the wavelength and intensity of the fluorescence spectrum of the light receiver that is incident upon changing the wavelength of the light source, and a memory that records the measured spectrum;
A fish fertilized egg inspection apparatus comprising a discriminator for discriminating pass / fail of the fertilized egg based on a predetermined spectral spectrum recorded in the memory The means for relatively horizontally moving the positions of the multi-well plate, the light source and the light receiver, and the same detection operation are sequentially repeated for the fertilized eggs in the adjacent wells, and the determination results obtained for each well are obtained. The fish fertilized egg inspection apparatus according to claim 7, further comprising a second memory for sequentially storing a pair of the two-dimensional address of each well.

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